Adeno-associated viral (AAV) liposomes and methods related thereto

ABSTRACT

A composition for genetic manipulation which comprises a liposome comprised of lipid material, and adeno-associated viral (AAV) material. Typically, the AAV material is plasmid, and comprises a terminal repeat of the AAV genome. Methods are disclosed for introducing genetic material into cells by use of AAV liposomes. Accordingly, genetic material was introduced and integrated into stem cells, T cells, primary tumor cells, or tumor cell lines.

This is a division of application Ser. No. 08/120,605 filed Sep. 13,1993, now abandoned.

TECHNICAL FIELD

The present invention involves cellular manipulation, more particularlyit relates to use of cationic liposomes to facilitate transfection byadeno-associated viral (AAV) plasmids.

BACKGROUND ART

Transfection of eukaryotic cells has become an increasingly importanttechnique for the study and development of gene therapy. Advances ingene therapy depend in large part upon the development of deliverysystems capable of efficiently introducing DNA into a target cell. Anumber of methods have been developed for the stable or transientexpression of heterologous genes in cultured cell types. These includetransduction techniques which use a carrier molecule or virus.

Most gene therapy strategies have relied on transduction by transgeneinsertion into retroviral or DNA virus vectors (Dimmock, N.J., "Initialstages in infection with animal viruses," J. Gen. Virol: (1982) 59:1-22;Duc-Nguyen, H., "Enhancing effect of diethylaminoethyl dextran on thefocus forming titer of a murine sarcoma virus (Harvey strain)," J.Virol. (1968) 2:643-644). However, adenovirus and other DNA viralvectors can produce infectious sequelae, can be immunogenic afterrepeated administrations, and can only package a limited amount ofinsert DNA.

Of the viral vector systems, the recombinant adeno-associated viral(AAV) transduction system has proven to be one of the most efficientvector systems for stably and efficiently carrying genes into a varietyof mammalian cell types (Lebkowski, J. S., et al., "Adeno-associatedvirus: A vector system for efficient introduction and integration of DNAinto a variety of mammalian cell types," Mol. Cell. Biol. (1988)3:3988-3996). It has been well-documented that AAV DNA integrates intocellular DNA as one to several tandem copies joined to cellular DNAthrough inverted terminal repeats (ITRs) of the viral DNA, and that thephysical structure of integrated AAV genomes suggest that viralinsertions usually appear as multiple copies with a tandem head to tailorientation via the AAV terminal repeats (Kotin, R. M., et al.,"Site-specific integration of adeno-associated virus," Proc. Natl. Acad.Sci. (1990) 87:2211-2215; Samulski, R. J., et al., "Targeted integrationof adeno-associated virus (AAV) into human chromosome 19," EMBO J.(1991) 10:3941-3950; Ashktorab, H. and A. Srivastara, "Identification ofnuclear proteins that specifically interact with adeno-associated virustype 2 inverted terminal repeat hairpin DNA," J. Virol. (1989)63:3034-3039; Im, D. S., and Muzyczka, N., "Factors that bind toadeno-associated virus terminal repeats," J. Virol. (1989) 63:3095-4104;Snyder, R. O., et al., "Evidence for covalent attachment of theadeno-associated virus (AAV) rep protein to the ends of the AAV genome,"J. Virol. (1990) 64:6204-6213). Thus, the AAV terminal repeats (ITRs)are an essential part of the AAV transduction system.

Although recombinant adeno-associated viral (AAV) vectors differ fromadenoviral vectors, the transgene DNA size limitation and packagingproperties are the same as with any other DNA viral vectors.

AAV is a linear single stranded DNA parvovirus, and requiresco-infection by a second unrelated virus in order to achieve productiveinfection. AAV carries two sets of functional genes: rep genes, whichare necessary for viral replication, and structural capsid protein genes(Hermonat, P. L., et al., "Genetics of adeno-associated virus: Isolationand preliminary characterization of adeno-associated type 2 mutants," J.Virol. (1984) 51:329-339; Tratschin, J. D., et al., "Genetic analysis ofadeno-associated virus: Properties of deletion mutants constructed invivo and evidence for an adeno-associated virus replication function,"J. Virol. (1984) 51:611-619). The rep and capsid genes of AAV can bereplaced by a desired DNA fragment to generate AAV plasmid DNA.Transcomplementation of rep and capsid genes are required to create arecombinant virus stock. Upon transduction using such virus stock, therecombinant virus uncoats in the nucleus and integrates into the hostgenome by its molecular ends (Kotin, R. M., et al., "Site-specificintegration of adeno-associated virus," Proc. Natl. Acad. Sci. (1990)87:2211-2215; Samulski, R. J., et al., "Targeted integration ofadeno-associated virus (AAV) into human chromosome 19," EMBO J. (1991)10:3941-3950).

Although extensive progress has been made, transduction techniquessuffer from variable efficiency, significant concern about possiblerecombination with endogenous virus, cellular toxicity and immunologichost response reactions. Thus, there is a need for non-viral DNAtransfection procedures.

Liposomes have been used to encapsulate and deliver a variety ofmaterials to cells, including nucleic acids and viral particles(regarding nucleic acids, see: Dimmock, N.J., "Initial stages ininfection with animal viruses," J. Gen. Virol: (1982) 59:1-22;Duc-Nguyen, H., "Enhancing effect of diethylaminoethyl dextran on thefocus forming titer of a murine sarcoma virus (Harvey strain)," J.Virol. (1968) 2:643-644); regarding viral particles, see: Felgner, P.L., et al., "A highly efficient, lipid-mediated DNA transfectionprocedure," Proc. Natl. Acad. Sci. USA (1987) 84:7413-7417; Faller, D.V. and D. Baltimore, "Liposome encapsulation of retrovirus allowsefficient superinfection of resistant cell lines," J. Virol. (1984)49:269-272; Wilson, T., et al., "Biological properties of poliovirusencapsulated in lipid vesicles: Antibody resistance and infectivity invirus resistant cells," Proc. Natl. Acad. Sci. USA (1977) 74:3471-3475).

Preformed liposomes that contain synthetic cationic lipids have beenshown to form very stable complexes with polyanionic DNA (Felgner, P.L., et al., "A highly efficient, lipid-mediated DNA transfectionprocedure," Proc. Natl. Acad. Sci. USA (1987) 84:7413-7417; Rose, J. K.,et al., "A new cationic liposome reagent mediating nearly quantitativetransfection of animal cells," Biotechniques (1991) 10:520-525).Cationic liposomes, liposomes comprising some cationic lipid, thatcontained a membrane fusion-promoting lipiddistearoyl-phosphatidyl-ethanolamine (DSPE) have efficiently transferredheterologous genes into eukaryotic cells (Felgner, P. L., et al., "Ahighly efficient, lipid-mediated DNA transfection procedure," Proc.Natl. Acad. Sci. USA (1987) 84:7413-7417; Rose, J. K., et al., "A newcationic liposome reagent mediating nearly quantitative transfection ofanimal cells," Biotechniques (1991) 10:520-525). Cationic liposomes canmediate high level cellular expression of transgenes, mRNA (Malone, R.,et al., "Cationic liposome mediated RNA transfection," Proc. Natl. Acad.Sci. USA (1989) 86:6077-6081), or transcription factors (Debs, R., etal., "Regulation of gene expression in vivo by liposome-based deliveryof a purified transcription factor," J. Biol. Chem. (1990)265:10189-10192), by delivering them into a wide variety of culturedcell lines noted in these citations.

Ecotropic and amphotropic packaged retroviral vectors have been shown toinfect cultured cells in the presence of cationic liposomes, such asLipofectin (BRL, Gaithersburg, Md.), and in the absence of specificreceptors (Innes, C. L., et al., "Cationic liposomes (Lipofectin)mediate retroviral infection in the absence of specific receptors," J.Virol. (1990) 64:957-961).

Overall, cationic liposomes have been shown to spontaneously complexwith plasmid DNA or RNA in solution; the liposome comprising nucleicacids then facilitates fusion with cells in culture, resulting in theefficient transfer of nucleic acids to a wide variety of eukaryotic celltypes. Liposome vectors are not subject to the DNA size and packagingproperties that limit recombinant AAV vectors and adenoviral vectors.Thus, viral infection has been enhanced by coating virus with cationicliposomes and efficiently delivering the virus into cells.

Even though non-viral techniques have overcome some of the problems ofthe viral systems, there remains a need for improved transfectionefficiency in non-viral systems (Hug, P. and R. G. Sleight, "Liposomesfor the transformation of eukaryotic cells," Biochem. Biophys. Acta(1991) 1097:1-22; Felgner, P. L., et al., "A highly efficient,lipid-mediated DNA transfection procedure," Proc. Natl. Acad. Sci. USA(1987) 84:7413-7417). To a certain extent, improved efficiency isattained by the use of promoter enhancer elements in the plasmid DNAconstructs (Philip, R., et al., "In vivo gene delivery: Efficienttransfection of T lymphocytes in adult mice,;" J. Biol. Chem. (1993)28:16087-16090).

DISCLOSURE OF INVENTION

Cationic liposomes were used to facilitate adeno-associated viral (AAV)plasmid transfections of primary and cultured cell types. AAV plasmidDNA, complexed with liposomes showed several-fold higher levels ofexpression than complexes with standard plasmids. In addition,expression lasted for a period of 30 days without any selection. AAVplasmid:liposome complexes induced levels of transgene expression thatwere comparable to those obtained by recombinant AAV transduction. Highlevel gene expression was observed in freshly isolated CD4⁺ and CD8⁺ Tcells, and CD34⁺ stem cells from normal human peripheral blood.

Primary breast, ovarian and lung tumor cells were transfected using theAAV plasmid DNA:liposome complexes. Transfected tumor cells were able toexpress transgene product after lethal irradiation. Transfectionefficiency ranged from 10-50% as assessed by β-galactosidase geneexpression. The ability to express transgenes in primary tumor cells isutilized to produce tumor vaccine and to produce lymphoid cells thatpermit highly specific modulations of the cellular immune response incancer and AIDS, and in gene therapies.

Thus, a composition for genetic manipulation is disclosed. Thecomposition comprises a liposome, itself comprising lipid material, andadeno-associated viral material. The lipid material may itself comprisecationic lipid material. The adeno-associated viral material cancomprise an inverted terminal-repeat of the AAV genome. The AAV materialcan be an AAV plasmid. Furthermore, the AAV material can have twoinverted terminal repeats and a promoter, such as a CMV immediate-earlyearly promoter, a CMV immediate-late promoter, an ADA promoter or a TKpromoter can be used. Additionally, a composition for geneticmanipulation in accordance with the invention can comprise geneticmaterial of interest, typically, transgene material. The transgenematerial can be placed between two inverted terminal repeats of the AAVplasmid. Cells transfected by a composition in accordance with theinvention are also disclosed.

Moreover, a method for introducing genetic material of interest into acell is disclosed. The method comprises steps of providing a liposomecomprising adeno-associated viral material and genetic material ofinterest. The AAV:liposome complex is then cultured with a host cell,whereby the genetic material of interest is introduced into the hostcell. The host cell can be CD34⁺ stem cells, T cells, primary tumorcells, or cells of a tumor cell line, such as rat bladder (MBT-2), or arat prostate cell line (R3327). Additionally, a method in accordancewith the invention can be used to integrate genetic material of interestinto the genetic material of a host cell.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Plasmid maps of three plasmids used in the present studies. Theplasmid pACMV-IL2 contained the CMV promoter, IL-2 cDNA and Ratpreproinsulin and SV40 polyadenylation sequences identical topBC12/CMV-IL2 plasmid, additionally pACMV-IL2 also had AAV invertedterminal repeats (ITRs) at both ends. The plasmid pA1CMVIX-CAT wasconstructed with CMV promoter and CAT gene inserted between the two AAVITRs.

FIGS. 2a-b. FIG. 2a depicts the levels of gene expression induced byplasmid DNA:liposome complexes. Various IL-2 plasmid constructs weretested for their capability to induce gene expression with a rat bladderand a rat prostate cell line, when the constructs were complexed withliposomes. In both cell lines, the AAV plasmid construct showed thehighest level of expression. The levels are expressed as picogram per mlper 10⁶ cells. FIG. 2b depicts the time-course of gene expressioninduced by AAV plasmid:liposome complexes. To compare the duration oftransgene expression, the prostate cell line was transfected with theAAV plasmid (pACMV-IL2) and the corresponding control plasmid(pBC12/CMV-IL2) complexed with liposomes. Supernatants were collected atvarious time points and assayed for IL-2 levels using an ELISA. IL-2levels are expressed as picogram/ml/10⁶ cells in 24 hrs of culture.

FIGS. 3a-b. A comparison of AAV plasmid:liposome complex mediatedtransfection to recombinant AAV transduction. To determine whether thelevels of gene expression induced by AAV plasmid:liposome complexes wereequivalent to rAAV transduction, the prostate cell line (FIG. 3a) andbladder line (FIG. 3b) were used to compare the transfection andtransduction of IL-2 gene. IL-2 levels were assessed using an ELISA. Thelevels are expressed as picogram/ml/10⁶ cells in 24 hrs of culture.

FIG. 4. Expression of IL-2 gene by lipofection with AAV plasmid:liposomecomplexes of various primary tumor cells. One lung, one ovarian, and twobreast tumor samples were isolated from fresh tumor biopsies. IL-2levels were measured using an ELISA. The levels are indicated aspicogram/ml/10⁶ cells in 24 hrs of culture.

FIGS. 5a-b. Expression of IL-2 by cells transfected in accordance withthe invention, then subjected to lethal irradiation. To determine theeffect of irradiation on gene expression, the prostate cell line (FIG.5a) and primary breast cells (FIG. 5b) were transfected and assessed forgene expression after lethal irradiation, as described herein.Supernatants were collected 24, 48, 72 and 96 hrs after irradiation andtested for IL-2 levels. IL-2 levels are expressed as pg/ml/10⁶ cells in24 hr culture.

FIG. 6. Efficiency of AAV:liposome transfection as measured by β-galgene expression. The β-gal reporter gene was used to assess thetransfection efficiency on a per cell basis. The prostate cell line wasused for transfection, as described herein. The data is represented aspercent of cells positive for fluorescence.

FIGS. 7a-d. Thin layer chromatography studies depicting transfected Tlymphocytes. Blood was obtained from donors referred to as A or B.Donor's A or B peripheral blood was used to isolate T cells, and fortransfection. Primary T cells freshly isolated from a donor's peripheralblood were tested for transgene expression using AAV plasmidDNA:liposome complexes. T lymphocytes were fractionated as CD3⁺ (FIG.7a), or CD5/8⁺ (FIG. 7b), or as CD4⁺ (FIG. 7c) or CD8⁺ (FIG. 7d)populations using AIS MicroCELLector devices. The relevant cells werecaptured and cultured as described herein. Thereafter, 5-10×10⁶ cellswere plated and transfected with 50 micrograms of AAV plasmid DNA and 50or 100 nmoles of liposomes to obtain 1:1 or 1:2 DNA:liposome ratios. Thecells were harvested 3 days after transfection. Normalized proteincontent from the extracts were assayed for CAT activity using achromatographic assay.

FIG. 8. Thin layer chromatography of peripheral blood CD34⁺ stem cellstransfected with AAV plasmid:liposomes. The cells were harvested on Day3 and Day 7 after transfection. Normalized protein content from theextracts were assayed for CAT activity using a chromatographic assay.

FIGS. 9a-b. Enhanced chemiluminescence (ECL) Southern analysis ofgenomic DNA from clones transfected with AAV plasmid DNA:liposomecomplexes. In FIG. 9a, samples were digested with Bam HI and Hind IIIand probed with IL-2. For the data in FIG. 9b, samples were digestedwith Bam HI and probed with IL-2. All clones analyzed show presence ofIL-2 gene, as demonstrated by the 0.685 kb bands. For FIGS. 9a-b:

lane 1: 1kb ladder

lane 2: plasmid cut with Bam HI/HindIII (9a) and BamHI/pvuII/pvuII (9b).

lane 3: R33 untransfected

lanes 4-11: clones

FIGS. 10a-b. Southern analysis (³² P) of clone 1A11 and 1B11. AfterSouthern blotting, the filter depicted in FIG. 10a was probed with a0.68 kb IL2 Bam HI/Hind III fragment of pACMV-IL-2. For FIG. 10a:

lane 1: clone 1A11 cut with Bam HI/Hind III

lane 2: clone 1B11 cut with Bam HI/Hind III

lane 3: clone R33 cut with Bam HI/Hind III

lane 4: clone 1A11 cut with Bam HI

lane 5: clone 1B11 cut with Bam HI

lane 6: clone R33 cut with Bam HI

lane 7: clone 1A11 cut with Hind III

lane 8: clone 1B11 cut with Hind III

lane 9: clone R33 cut with Hind III

lane 10: left empty

lane 11: pACMV-IL2 plasmid cut with Bam HI/Hind III

lane 12: pACMV-IL2 plasmid cut with Hind III/pvuII

lane 13: pACMV-IL2 plasmid cut with Bam HI/pvuII

For the data shown in FIG. 10b, the filter was probed with a 0.85 kbpvuII/HindIII (AAV ITR/CMV) fragment of the plasmid pACMV-IL2. For FIG.10b:

lane 1: clone 1A11 cut with smaI

lane 2: clone 1B11 cut with smaI

lane 3: clone R33 cut with smaI

lane 4: clone 1A11 cut with pvuII/Hind III

lane 5: clone 1B11, cut with pvuII/Hind III

lane 6: clone R33, cut with pvuII/Hind III

lane 7: pACMV-IL2, cut with Bam HI/Hind III

lane 8: pACMV-IL2, cut with Hind III/pvuII

lane 9: pACMV-IL2, cut with smaI

lane 10: 1kb ladder

MODES FOR CARRYING OUT THE INVENTION

The studies, disclosed for the first time herein, examined thetransportation into cells of AAV plasmid DNA by a system that does notinvolve viral transduction. Alternatively, a method in accordance withthe invention efficiently transfected several mammalian cell types byuse of liposomes comprising AAV material. The present invention relatesto transfection, and utilizes the elegant carrier system of lipofectiontogether with the proficient transduction capability of the AAV plasmidconstruct. Advantageously, cationic liposomes were used as a means tofacilitate the entry of AAV plasmid DNA into cells in the absence of repand capsid transcomplementation, recombinant virus or wild type AAV. Alipofection method in accordance with the invention was evaluated toassess the efficiency of gene expression. The present data establishedthe ability to transfect unmodified stem cells, unmodified primarylymphoid cells such as T cells, a variety of freshly isolated tumorcells, and cultured mammalian cell types, with high efficiency for bothtransient and sustained expression of DNA. The ability to efficientlytransfect unmodified T cells and unmodified stem cells is disclosed forthe first time in the art.

I. SOURCE MATERIALS AND METHODS EMPLOYED

A. Cell Lines.

A rat prostate cell line (R3327) and rat bladder cell line (MBT-2) wereobtained from Dr. Eli Gilboa, Duke University. Both cell lines weremaintained in RPMI-1640 medium supplemented with 5% fetal bovine serum(FBS). Cell line 293 is a human embryonic kidney cell line that wastransformed by adenovirus type 5, and was obtained from the ATCC(Graham, F. L., et al., "Characteristics of a human cell linetransformed by DNA from human adenovirus type 5," J. Gen. Virol. (1977)3:59-72). Cell line 293 was grown in Dulbecco modified eagle mediumsupplemented with 10% FBS.

B. Cell preparation of Primary Tumor Cells.

Primary lung, ovarian and three breast tumor cells were obtained fromsolid tumors of patients. The tumor samples were minced into smallpieces and digested in 200 ml of AIM V medium (Gibco), supplemented with450 u/ml collagenase IV (Sigma), 10.8K units/ml DNase I (Sigma), and2000 u/ml hyaluronidase V (Sigma) (Topolian, S. L., et al., "Expansionof human tumor infiltrating lymphocytes for use in immunotherapytrials," J. Immunol. Methods (1987) 102:127-141). After 1-2 hours ofdigestion, cells were homogenized with a glass homogenizer (Bellco). Thecells were washed three times in DPBS-CMF (Whittaker). Lymphocytes wereseparated from non-lymphoid cells by capture on an AISMicroCELLector-CD5/8 device (AIS, Santa Clara, Calif.). Nonadherentcells (mainly tumor cells) were removed and cultured in RPMI 1640supplemented with 2 mM L glutamine, 100 u/ml penicillin-streptomycin,and 10% FBS. Tumor cells were cultured for 2 to 4 weeks prior totransfection.

C. Preparation of Peripheral Blood Mononuclear Cells.

Peripheral blood mononuclear cells (PBMCs) from healthy control patientswere isolated from buffy coats (Stanford University Blood Bank,Stanford, Calif.), using Lymphoprep (Nycomed, Norway).

T cells or T cell subsets were further isolated using AISMicroCELLectors (Applied Immune Sciences, Santa Clara, Calif.). Briefly,PBMCs were resuspended at 15×10⁶ cells per ml in 0.5% Gamimmune (Miles,Inc., Elkhart, Ind.) and loaded onto washed CD3, CD4, CD8, CD5/8, orCD34 AIS MicroCELLectors. After 1 hour, nonadherent cells were removedfrom the AIS MicroCELLectors. Complete medium, RPMI 1640 (Whittaker)containing 10% fetal bovine serum, 2 mM L-glutamine, and 100 u/mlpenicillin/streptomycin was added to the adherent cells in the AISMicroCELLectors. After 2-3 days in a 5% CO₂, 37° C. humidifiedenvironment, adherent cells were removed and prepared for transfection.

D. Plasmid Preparation.

A first study plasmid (pACMV-IL2) was used in the present studies; thisplasmid contained the human interleukin-2 gene (IL-2) as IL-2 cDNA, andthe immediate-early promoter-enhancer element of the humancytomegalovirus (CMV), and Rat preproinsulin and SV40 polyadenylationsequences, flanked by adeno-associated virus inverted terminal repeats(ITRs) at both ends. (This plasmid was provided by Dr. J. Rosenblatt,UCLA, CA; Dr. Rosenblatt's name for the plasmid is pSSV9/CMV-IL2). Acorresponding control plasmid pBC12/CMV-IL2, which was identical topACMV-IL2 but which lacked the AAV terminal repeats, was also used (FIG.1).

A second study plasmid, pA1CMVIX-CAT, contained the CMV immediate-earlypromoter enhancer sequences, and an intron derived from pOG44(Strategene); the bacterial CAT gene; SV40 late polyadenylation signalflanked by AAV terminal repeats in a pBR322 backbone (FIG. 1).

The plasmids pATK-βgal and pAADA-βgal contained the βgal gene linked toeither the TK or ADA promoter, respectively, in an AAV plasmid backbone.(βgal plasmids provided by Dr. Eli Gilboa, Duke Univ.) Standard plasmidconstructs that contained the IL-2 gene, but that did not contain AAVcomponents were also used. The standard plasmid constructs carried theIL-2 gene, with an adenosine deaminase (ADA), a thymidine kinase (TK) orthe immediate-late cytomegalovirus (CMV) promoter. (standard plasmidsobtained from ATCC)

                  TABLE 1    ______________________________________    Plasmids Used in Present Studies    Plasmid Name               Promoter        Genomic Elements    ______________________________________    pACMV-IL2  CMV (immediate-early)                               IL2, AAV    pBC12/CMV-IL2               CMV (immediate-early)                               IL2    pA1CMVIX-CAT               CMV (immediate-early)                               CAT, AAV    pADA-IL2   ADA             IL2    pTK-IL2    TK              IL2    pCMV-IL2   CMV (immediate-late)                               IL2    pATC-βgal               TK              βgal, AAV    pAADA-βgal               ADA             βgal, AAV    ______________________________________

All plasmids were isolated by alkaline lysis and ammonium acetateprecipitation, followed by treatment with DNase-free RNase,phenol/chloroform/isoamyl extractions and ammonium acetate precipitation(Ausubel, F. M., et al., Current Protocols in Molecular Biology (JohnWiley and Sons, Inc. 1993)).

E. Liposome Preparation.

Small unilamellar liposomes were prepared from the cationic lipiddioctadecyl-dimethyl-ammonium-bromide (DDAB) (Sigma) in combination withthe neutral lipid dioleoyl-phosphatidyl-ethanolamine (DOPE) (AvantiPolar Lipids). Lipids were dissolved in chloroform. DDAB was mixed withDOPE in either a 1:1 or 1:2 molar ratio in a round-bottomed flask, andthe lipid mixture was dried on a rotary evaporator. The lipid film wasrehydrated by adding sterile double distilled water to yield a finalconcentration of 1 mM DDAB. This solution was sonicated in a bathsonicator (Laboratory Supplies, Hicksville, N.Y.) until clear. Theliposomes were stored at 4° C. under argon. For in vivo use of liposomesvia intravenous administration a DDAB:DOPE ratio of 1:4 to 1:5 is used;for intraperitoneal administration a DDAB:DOPE ratio of 1:1 to 1:2 isused.

F. Preparation of recombinant AAV (rAAV) for transduction.

For the preparation of recombinant AAV stocks, cells of the cell line293 were split and grown to approximately 30-50% confluence. Thereupon,the cells were infected with adenovirus type 5 at a multiplicity ofinfection of 1 to 5, and incubated at 37° C. After 2 to 4 hours, theinfected cells were cotransfected with 10 μg of a plasmid comprising agene of interest and 10 μg of the rep capsid complementation plasmid,pΔBal, per 100 mm tissue culture dish (0.5-1×10⁷ cells). Calciumphosphate coprecipitation was used for transfection (Hermonat, P. L. andMuzyczka, N., "Use of adeno-associated virus as a mammalian DNA cloningvector. Transduction of neomycin resistance into mammalian tissueculture cells," Proc. Natl. Acad. Sci. USA (1984) 81:6466-6470). At 12to 18 hours after transfection, the medium was removed from the cellsand replaced with 5 ml of DMEM medium containing 10% FBS.

At 48 to 72 hours after transfection, AAV was harvested according to thefollowing procedure: Cells and medium were collected together, andfreeze thawed three times to lyse the cells. The suspension of cells andmedium was then centrifuged to remove cellular debris, and thesupernatant was incubated at 56° C. for 1 hour to inactivate adenovirus(Hermonat, P. L. and N. Muzyczka, "Use of adeno-associated virus as amammalian DNA cloning vector. Transduction of neomycin resistance intomammalian tissue culture cells," Proc. Natl. Acad. Sci. USA (1984)81:6466-6470; Tratschin, J. D., et al., "Adeno-associated virus vectorfor high frequency integration, expression, and rescue of genes inmammalian cells," Mol. Cell. Biol. (1985) 5:3251-3260). After heatinactivation, the viral supernatant was filtered through celluloseacetate filters (1.2 μm). Viral stocks were then stored at -20° C. Onemilliliter of AAV supernatant was used to transduce 1×10⁶ cells.

G. Cellular Transfection "Lipofection".

For primary tumor cells and both rat tumor cell lines (R3327 and MBT-2),1×10⁶ cells were plated in 2 ml serum-free medium per well of a 6 welldish. Thereafter, 5 μg of AAV plasmid DNA was mixed with 5 nmoles ofDDAB as liposomes composed of DDAB and DOPE in a 1:2 molar ratio,respectively. Serum-free medium (0.5 ml) was added to the AAV:liposomecomplex, which was then transferred to the cells. To effect lipofection,the cells were incubated at room temperature for 5 minutes, then fetalbovine serum was added to the cells to yield a final concentration of 5%fetal bovine serum.

For T cells, 5-10×10⁶ cells were plated in 1 ml of serum-free medium perwell of a 6 well dish. 50 μg of plasmid DNA was mixed with 50 nmoles ofDDAB as liposomes composed of DDAB and DOPE in a 1:1 molar ratio. Thetransfections "lipofections" were then performed as above.

For stem cells, 1-2×10⁶ cells were transfected with complexes comprising10 micrograms of plasmid DNA and 10 nmoles of liposome. The transfectedcells were cultured with medium containing stem cell factor, IL-3 andIL-1. On Day 3 and 7, the cells were harvested and extracts were made.

H. IL-2 Assay.

Cells were counted, and 1×10⁶ cells were plated in 1 ml per well of a 24well plate. The following day, supernatants were collected and assessedby using a Quantikine IL-2 ELISA kit (R&D Systems, Minneapolis, Minn.).IL-2 levels were defined as picograms/ml of the supernatant.

I. β-galactosidase Assay.

The FluoReporter lacZ gene fusion detection kit from Molecular Probes(Eugene, Oreg.) was used to quantitate lacZ β-D-galactosidase in singlecells by measurement of the fluorescence of the enzyme hydrolysisproduct, fluorescein. The AAV/β-gal plasmids (pATK-βgal and pAADA-βgal)were used with this kit. Fluorescein is produced by enzymatic cleavageof fluorescein di-b-D-galactopyranoside (FDG) in cells that express themarker gene b-D-galactosidase. The cells then were analyzed using flowcytometry (FACScan, Becton Dickinson, San Jose, Calif.)

II. STUDY RESULTS

A. Level of IL-2 gene expression by use of AAV plasmid:cationic liposomecomplex.

To evaluate the gene transfer efficiency of AAV plasmids, the IL-2 genetransfer efficiency of AAV plasmids were compared to the efficiencies ofstandard plasmid constructs. The standard plasmids carried the IL-2gene, with an adenosine deaminase (ADA) promoter (pADA-IL2), a thymidinekinase (TK) promoter (pTKIL-2), or the immediate-late cytomegalovirus(CMV) promoter (pCMV-IL2). The AAV IL-2 study plasmid (pACMV-IL2)contained the CMV promoter (immediate early), with the IL-2 gene placeddownstream of the promoter. (FIG. 1) As shown in FIG. 1, thecorresponding control plasmid, the pBC12/CMV-IL2 construct, wasidentical to pACMV-IL2, but lacked the AAV terminal repeats (ITRs).

All five plasmids containing the IL-2 gene were complexed with liposomesand tested for transfection efficiency on the two cultured tumor celllines: the rat bladder (MBT-2) and the rat prostate (R3327) cell lines.The cell lines were transfected with 10 micrograms of plasmid DNAcomplexed to 10 nmoles of liposomes per 1×10⁶ cells. Supernatants werecollected on Day 3 and tested for the levels of IL-2 using an IL-2 ELISAkit.

The AAV plasmid (pACMV-IL2) induced the highest levels of expression inboth cell lines (FIG. 2a). The IL-2 gene with an ADA promoter (pADA-IL2)induced the least amount of expression in both cell lines. As shown inFIG. 2a, both TK and CMV (immediate-late promoter) IL-2 constructsinduced comparable levels of IL-2 expression in both cell lines.However, the pBC12/CMV-IL2 plasmid, which contained CMV immediate-earlypromoter showed higher levels of gene expression in the prostate cellline when compared to the bladder cell line. Among the plasmids tested,the AAV IL-2 study plasmid induced the highest level of expression inboth cell lines, with a significant level of increase observed in theprostate cell line.

As illustrated in FIG. 2b, the duration of expression induced by thecorresponding control plasmid (pBC12/CMV-IL2) and the AAV IL-2 studyplasmid (pACMV-IL2) in the prostate cell line R3327 were studied.Expression was assessed up to 30 days in these cultures without anyselection. The cells were seeded at 1×10⁶ /ml and supernatants werecollected for analysis every 24 hours. The cells doubled every 48 hoursin culture. The data in FIG. 2b indicate that, in addition to theenhanced levels of expression, the duration of expression lasted 30 dayspost-transfection with AAV plasmid (pACMV-IL2). Notably, significantexpression continued throughout the full duration of the time period ofevaluation. As shown in FIG. 2b, both plasmids induced maximum levels ofexpression between Day 2 and Day 7, by Day 15 IL-2 levels declined andthen were maintained at approximately 100 pg/ml only in the AAV plasmidtransfected group. Similar sustained levels of expression were observedin the bladder cell line, as well as with cells from a primary lungtumor, when AAV plasmid:liposome complexes were used for transfection(data not illustrated).

B. Comparison of AAV plasmid:liposome transfection "lipofection" andrecombinant AAV transduction.

The prostate and bladder cell lines were transfected and transduced, todetermine whether optimal AAV:liposome transfection was comparable tooptimal recombinant AAV transduction. For optimal transfection, 10micrograms of AAV plasmid DNA was complexed to 10 nmoles of liposomesper 1×10⁶ cells in 2 ml final volume. For maximal rAAV transduction, 2ml of the viral supernatant was added to 1×10⁶ cells in 1 ml of completemedium. After 24 hrs, the cells were washed and resuspended in freshcomplete medium. Supernatant was collected at various time points aftertransfection and transduction.

In the prostate line (FIG. 3a), transfection induced higher levels ofexpression than AAV transduction under test conditions (2 ml of viralsupernatant for 1×10⁶ cells, versus 10 μg DNA:10 nmoles of liposomes).Although results on Day 3 through Day 5 showed approximately 10-foldhigher levels of IL-2 with transfection, by Day 20 comparable levelswere observed in both transfected and transduced groups.

Transduction-with recombinant AAV initially induced higher levels ofIL-2 production in the bladder cell line, as compared to transfectionusing liposomes (FIG. 3b). Similar to the prostate cell line, thebladder cell line also showed a decline in IL-2 levels by Day 20, withcomparable levels of IL-2 produced through Day 33 in both transfectedand transduced groups.

C. Transfection of primary tumor cells using AAV plasmid DNA:liposomecomplexes.

In the foregoing experiments disclosed herein, significant transgeneexpression was demonstrated in cultured cell lines. In order to assesswhether cationic liposome:AAV plasmid DNA complexes also mediatedcomparable transgene expression in freshly isolated primary tumor cells,cells of four different primary tumors were transfected with the AAVIL-2 study plasmid using liposomes. Tumor cells were cultured inRPMI-1640 medium supplemented with 10% FBS for 2-3 weeks prior to thetransfection. The cells were plated to 1×10⁶ cells per ml concentrationand transfected with 10 micrograms of DNA complexed with 10 nmoles ofliposomes. Supernatants were collected on Day 2 and 3.

As shown in FIG. 4, all four primary cell types produced significantlevels of IL-2 after transfection. The highest level of expression wasobserved on Day 3 during the 10 Day study period (lung and one breastsample were studied for longer periods). IL-2 gene expression wasfollowed in cells of the lung tumor and in cells of one of the breasttumors as long as 25 days after transfection in culture. The levels onDay 15 were equivalent (100 pg/ml IL-2) in both cell lines, and thecells derived from primary tumors. (data not shown)

D. Effect of lethal irradiation on transgene expression.

To determine the effect of irradiation on gene expression, the prostatecell line (FIG. 5a) and cells of a primary breast tumor (FIG. 5b) weretransfected and assessed for gene expression after lethal irradiation.Both cell types were transfected using optimal AAV plasmid:liposomecomplexes. On the second day after transfection, an aliquot of eachculture was subjected to 6000 rad using ⁶⁰ Co irradiator, wherebycellular division is abolished, and the aliquots were then kept inculture. One-half of each culture was maintained as a non-irradiatedcontrol. The aliquots were subjected to 6000 rad using a ⁶⁰ COirradiator, while the expression level of IL-2 was approximately 300-400pg/ml. Supernatants were collected 24, 48, 72 and 96 hrs afterirradiation, and then tested for IL-2 levels.

As shown in FIGS. 5a-b, lethal irradiation post-transfection did notinhibit transgene expression. Neither the prostate cell line nor theprimary tumor cells exhibited any change in IL-2 expression afterirradiation. Thus, although cellular division was abolished, IL-2secretion was not sensitive to irradiation. This is advantageous, sincemost gene therapy strategies involve gene delivery to primary Tlymphocytes, which do not generally divide absent modification, or totumor cells, as discussed in greater detail below.

E. Level of β-D-galactosidase gene expression by use of AAVplasmid:liposome complex.

To demonstrate the expression levels on a per cell basis, theβ-D-galactosidase gene was used for transfection experiments. Each oftwo AAV β-gal plasmids (pATK-Bgal and pAADA-Bgal) (plasmids obtainedfrom Dr. Eli Gilboa, Duke University) were complexed with cationicliposomes and used for transfection of the prostate cell line. Tenmicrograms of pATK-βgal or pAADA-βgal plasmid DNA was complexed with 10nmoles of liposomes, the complexes were then used to transfect 1×10⁶cells in 2 ml volume. At various time points, approximately 5×10⁵ cellswere harvested and stained with fluorescent substrate FDG and analyzedusing flow cytometry.

Maximum transgene expression was observed between Day 7 and Day 15 (FIG.6). Significant levels of β-gal activity were observed through Day 25.Flow cytometry analysis of β-gal positive cells showed maximal levels of10 to 50% transfection efficiency with both plasmid constructs. Thelevels declined to 5 to 10% by Day 25. The expression pattern andduration was similar to that of IL-2 expression set forth above.

F. Transgene expression induced by AAV plasmid:liposome complex infreshly isolated peripheral blood T cell subpodulation.

The effect of AAV plasmid:liposome complex in transfecting freshlyisolated human peripheral blood T cell populations was examined. Thegene for chloramphenicol acetyl transferase (CAT) enzyme was used as thereporter gene in the pA1CMVIX-CAT plasmid (FIG. 1). The pA1CMVIX-CATconstructs were made using the AAV backbone (pA1) with CMVimmediate-early promoter enhancer sequences and CAT gene. Total andpurified CD4⁺ and CD8⁺ subpopulations of T cells were used fortransfections. Both total (CD3 or CD5/8 selected) and purified (CD4 orCD8 selected) subpopulations of T cells (FIGS. 7a-d), as well as CD34⁺stem cells (FIG. 8), described in Section G. below, showed significantlevels of CAT gene expression.

Primary T cells freshly isolated from peripheral blood were tested fortransgene expression using AAV plasmid DNA:liposome complexes. Resultsof thin layer chromatography assays for CAT activity from CD3⁺ T cells,CD5/8 selected T cells (total T cells), the CD4⁺ subpopulation of Tcells, and the CD8⁺ subpopulation of T cells are depicted in FIGS. 7a-d,respectively.

T lymphocytes were fractionated as CD3⁺, or CD5/8⁺ or CD4⁺ or CD8⁺populations using AIS MicroCELLector devices. The relevant cells werecaptured and nonadherent cells were washed off. The adherent cells wereremoved from the devices after 2 days in culture with RPMI-1640 and 10%FBS. Five to 10×10⁶ cells were plated and transfected with 50 microgramsof AAV plasmid DNA and 50 or 100 nmoles of liposomes to obtain 1:1 or1:2 DNA:liposome ratios. The cells were harvested 3 days aftertransfection and the cell extracts normalized by protein content and CATactivity measured using a chromatographic assay. Blood was obtained fromDonors referred to as A or B. Peripheral blood of Donor A or B was usedto isolate the T cells, and for transfection.

As depicted in FIGS. 7a-d, the lipid composition of the liposomescomprising AAV was varied, as was the ratio of DNA to liposome. In thestudy of CD3⁺ T cells (FIG. 7a) cells from one donor (Donor A) wereemployed. For the studies of CD5/8 selected T cells (FIG. 7b), the CD4⁺subpopulation of T cells (FIG. 7c), the CD8⁺ subpopulation of T cells(FIG. 7d), and CD34⁺ stem cells (FIG. 8), described below, cells derivedfrom two patients. (Donor A and Donor B) were utilized.

                  TABLE 2    ______________________________________    Conditions Employed for Studies Depicted in FIG. 7a    Condition Number                  Parameters    ______________________________________    1.            pA1CMVIX-CAT + DDAB:DOPE (1:1),                  DNA:liposome ratio (1:1).    2.            pA1CMVIX-CAT + DDAB:DOPE (1:1),                  DNA:liposome ratio (1:2).    3.            pA1CMVIX-CAT + DDAB:DOPE (1:2),                  DNA:liposome ratio (1:1).    4.            pA1CMVIX-CAT + DDAB:DOPE (1:2),                  DNA:liposome ratio (1:2).    ______________________________________

                  TABLE 3    ______________________________________    Conditions Employed for Studies Depicted in FIG. 7b-d    Condition Number                  Parameters    ______________________________________    1.            pA1CMVIX-CAT + DDAB:DOPE (1:1),                  DNA:liposome ratio (1:1).    2.            pA1CMVIX-CAT + DDAB:DOPE (1:1),                  DNA:liposome ratio (1:2).    3.            pA1CMVIX-CAT + DDAB:chol (1:1),                  DNA:liposome ratio (1:1).    4.            pA1CMVIX-CAT + DDAB:chol (1:1),                  DNA:liposome ratio (1:2).    ______________________________________

For the studies depicted in FIGS. 7a-d, maximum levels of expressionwere observed on Days 2 and 3 in both total and purified subpopulations.Significant levels of expression were detected up to Day 14. The cellswere harvested 3 days after transfection, and normalized protein contentfrom each extract was analyzed for CAT activity. The same composition ofliposome, and the DNA to liposome ratio induced similar levels ofexpression in all the populations.

G. Transgene expression induced by AAV plasmid:liposome complex infreshly isolated CD34⁺ stem cells.

The effect of AAV plasmid:liposome complex in transfecting freshlyisolated human peripheral blood CD34⁺ stem cells was examined. The genefor chloramphenicol acetyl transferase (CAT) enzyme was used as thereporter gene in the pA1CMVIX-CAT plasmid (FIG. 1). The pA1CMVIX-CATconstructs were made, as described above. The level of CAT expression asdetermined by thin layer chromatography from CD34⁺ stem cells is setforth in FIG. 8.

                  TABLE 4    ______________________________________    Conditions Employed for Studies Depicted in FIG. 8    Condition Number                  Parameters    ______________________________________    1.            pA1 CMV IX CAT + DDAB:DOPE (1:1)                  1:1 DNA:liposome ratio.    2.            pA1 CMV IX CAT + DDAB:DOPE (1:1)                  1:2 DNA:liposome ratio.    ______________________________________

Freshly isolated CD34⁺ peripheral blood stem cells were transfected withAAV CAT plasmid DNA:liposome complexes. CD34⁺ cells were purified fromperipheral blood using AIS CD34 MicroCELLectors after removingessentially all the T cells using CD5/8 MicroCELLector device. The stemcells were removed from the device and 0.5-1×10⁶ cells were transfectedwith complexes comprising 10 micrograms of plasmid DNA and 10 nmoles ofliposome. The transfected cells were cultured with medium containingstem cell factor, IL-3 and IL-1. On Day 3 and 7, the cells wereharvested and extracts were made. Normalized protein content from theextract was assayed for CAT activity. As shown in FIG. 8., there weresignificant levels of CAT gene expression in the CD34⁺ peripheral bloodstem cells.

H. Integration Studies.

FIGS. 9a-b illustrates enhanced chemiluminescence (ECL) Southernanalyses of genomic DNA from stable clones (clones stable at leastbeyond Day 30) that were transfected with AAV plasmid DNA:liposomecomplexes in accordance with the invention. Genomic DNA was isolated andanalyzed using the ECL direct nucleic acid labelling and detectionsystem (Amersham). IL-2 probe was prepared from the 0.685 kb IL-2 genefrom pACMV-IL2. After hybridization, the membrane was washed twice in0.5x SSC/0.4% SDS at 55° C. for 10 minutes and twice in 2x SSC at roomtemperature for 5 minutes.

In FIG. 9a, samples were digested with Bam HI and Hind III and probedwith IL-2. As shown in FIG. 9a, all clones showed the presence of theIL-2 gene, as demonstrated by the 0.685 kb band in Bam HI and Hind IIIdigested genomic DNA.

For the data in FIG. 9b, samples were digested with Bam HI and probedwith IL-2. Again, all clones showed IL-2 gene integration. (FIG. 9b) InFIG. 9b, integration of IL-2 was demonstrated by the high molecularweight bands (between 1.6 and 2 kb), bands which are consistent withintegration of the gene in conjunction with attached host genomicmaterial obtained via digestion. The data in FIG. 9b indicate that therewas more than one integration site, since there were multiple highmolecular weight bands in the Bam HI digested genomic DNA. Furthermore,the integration site was shown to be in different locations in differentclones, as demonstrated by the different size bands in the digestedclones (FIG. 9b).

FIGS. 10a-b depict chromosomal DNA analyses, using a ³² P Southernassay, of two clones obtained from the present study. Nuclear DNA wasisolated from the two IL-2 clones (1A11 and 1B11) using the Hirtfractionation protocol. As a negative control, total DNA was isolatedfrom untransfected cells of the R3327 cell line. After restrictionenzyme digestion, 10 micrograms of each sample, along with appropriateplasmid controls, were loaded onto a 1% agarose gel, electrophoresed,denatured and transferred onto Hybond+ membrane. The filters werehybridized overnight at 68° C. with DNA fragments labelled with ³² P byrandom priming. The membranes were then washed at 68° C. for 2x 30minutes each with 2x SSC, 0.1% SDS and 0.2x SSC, 0.1% SDS.Autoradiograms of these filters were exposed on x-ray film.

In FIG. 10a, the IL-2 gene was again used as the probe. Thus, afterSouthern blotting, the filter depicted in FIG. 10a was probed with a0.68 kb IL2 Bam HI/Hind III fragment of pACMV-IL2. The data in FIG. 10aindicate that the number of copies of the IL-2 gene that integrated intoa clone, varied from clone to clone; this finding was demonstrated bythe various densities of the 0.685 kb band in the digests (as specifiedin the Brief Description of the Drawings) of cells of the two clones.Moreover, higher molecular weight bands were also demonstrated, which isconsistent with integration of the IL-2 gene, together with host genomicmaterial obtained from the various digest protocols.

For the data shown in FIG. 10b, the filter was probed with a 0.85 kbpvuII/HindIII (AAV ITR/CMV) fragment of the plasmid pACMV-IL2. The datain FIG. 10b indicate the presence of the right AAV ITR, as demonstratedby the 0.8 kb band in the smaI and pvuII digested chromosomal DNA. Thepresence of the left AAV ITR in one clone (clone A) was demonstrated bythe 2.1 kb band in the smaI and pvuII digested chromosomal DNA.

III. EXAMPLES

A method in accordance with the invention, utilizing liposomes thatcomprise AAV viral material, is used to deliver genes for cytokines,costimulatory molecules such as B7, and molecules having MHC class Iantigens into a wide variety of cell types. For example, such genomicmaterial can be delivered into primary tumor cells, for tumor vaccines.

A method in accordance with the invention, comprising use of liposomesthat contain AAV viral material, is used to deliver and express genesfor substances such as peptides, anti-sense oligonucleotides, and RNA.Upon expression of such peptides, anti-sense oligonucleotides and RNA, asubject's immune response is modulated. The modulation of the immuneresponse is either that of inducing the immune response or inhibitingthe immune response. Accordingly, HIV infection is treated by usinganti-sense oligonucleotides, RNA, or ribozymes that have been expressedby a method in accordance with the invention. Additionally, theimmunologic response to a tumor is modulated by use of peptides or RNAexpressed in accordance with the invention. A patient's immune responseis modulated so as to respond to tumor-specific and/or tumor-associatedantigens. Accordingly, non-immunogenic tumors are modified intoimmunogenic tumors which induce a cytolytic T cell response, both invivo and in vitro.

A method in accordance with the invention is used to deliver genes toprimary lymphoid cells, such as B cells or T cells. An alternate methodin accordance with the invention is used to deliver genetic material toCD34⁺ stem cells. Accordingly, the genes are expressed and are used intherapy for conditions such as HIV infection, conditions of geneticdefect, neoplasias, and auto-immune conditions, wherein expression agene of interest is desired, as is appreciated by one of ordinary skillin the art. For example, for a malignant neoplastic condition, the MDR Igene is delivered in accordance with the invention, is expressed, andhas therapeutic effect.

In a further example, CD8⁺ cells are selected with AIS MicroCELLectors.The source material for the CD8⁺ cells is peripheral blood for HIVpatients, and tumor samples for patients with neoplasias. The T cellsare then activated according to methods known in the art, such as by useof phytohemagglutinin (PHA). The activated cells are grown for 20 days.Thereafter, the cells are transfected in accordance with the inventionwith AAV:liposome complexes comprising IL-2 genomic material. Thetransfected cells are returned to the patient. Thus, the subsequentadministration of IL-2 to a patient in order to maintain their cytotoxicT cell activity is reduced. Advantageously, the IL-2 gene, administeredin accordance with the invention, permits lessened amounts of IL-2 to beprovided systemically to a patient. Reducing the amount of IL-2 that issystemically administered is advantageous, since IL-2 displayspotentially lethal dose-related toxicity.

IV. CONCLUSIONS

In the present studies, the AAV plasmid which contained transgene andAAV terminal repeats was used as a DNA vector, and cationic liposomeswere used as carrier molecules. It was demonstrated that the AAV plasmidDNA:liposome complexes efficiently transfected primary tumor cells,cultured cell lines, primary lymphoid cells, and CD34⁺ stem cells. Itwas also demonstrated that, in the absence of any recombinant virus(producible from rep and cap capsid particles in adenoviral infectedcells), integration with high level and sustained expression oftransgene was achieved by the elegant transfection process.

In addition to high levels of expression, the combination of AAVplasmid:liposomes disclosed herein induced long-term (up to 30 days)expression of genes (FIGS. 3a and 3b), in contrast to the transientexpression demonstrated by typical liposome-mediated transfection.Notably, sustained expression was demonstrated in the AAV plasmidlipofected group, as well as in the recombinant AAV transduced group(FIGS. 3a and 3b). Moreover, ten-fold higher levels of expression wereobserved with AAV plasmid as compared to standard plasmid transfection,as shown in FIGS. 2a and 2b.

Under the test conditions disclosed herein, there was no difference inefficiency between optimal AAV transduction and maximal AAVplasmid:liposome transfection. Concerning the time-course of expression,cationic liposomes had previously been shown to mediate only transientexpression of standard plasmid DNA in mammalian cell types (Felgner, P.L., et al., "A highly efficient, lipid-mediated DNA transfectionprocedure," Proc. Natl. Acad. Sci. USA (1987) 84:7413-7417; Rose, J. K.,et al., "A new cationic liposome reagent mediating nearly quantitativetransfection of animal cells," Biotechniques (1991) 10:520-525).Moreover, concerning the efficiency of integration, much lowerefficiency of integration into the host genome was observed in formerliposome-mediated transfection as compared to the results disclosedherein (Shaefer-Ridder, M., et al., "Liposomes as gene carriers:Efficient transfection of mouse L cells by thymidine kinase gene,"Science (1982) 215:166-168). As shown herein, however, cationicliposomes complexed with AAV plasmid DNA carrying the AAV terminalrepeats increased the genomic DNA integration, relative to the standardplasmid that lacked only the AAV terminal repeats (ITRs). Liposomescomprising AAV plasmid material delivered the plasmid DNA in the absenceof any specific cell surface receptors, and replaced the function ofvirus in gene delivery.

In the present studies, it was demonstrated that virus vectors can bealtogether replaced by liposomes, and efficient expression andintegration was attained by utilizing the construct, including the viralelements responsible for both the efficiency and integration. In thismanner, production of virus for infection can be avoided, and there isno possibility of an adverse recombinant event. The end results wereaccomplished by use of an elegant transfection process combining AAVplasmid and cationic liposomes.

In a preferred embodiment, the combination of AAV plasmid and cationicliposomes not only transfected the cultured cell lines efficiently, butalso transfected primary tumor cells and peripheral blood cells such asT cells and stem cells. These data are noteworthy since most genetherapy strategies involve gene delivery to primary T lymphocytes ortumor cells. Previously, these strategies have primarily relied upontransgene insertion into retroviral or DNA virus vectors (Dimmock, N.J.,"Initial stages in infection with animal viruses," J. Gen. Virol: (1982)59:1-22; Duc-Nguyen, H., "Enhancing effect of diethylaminoethyl dextranon the focus forming titer of a murine sarcoma virus (Harvey strain),"J. Virol. (1968) 2:643-644). A fundamental disadvantage of theretroviral system is understood to be the inability to transfectnon-dividing primary cells (Innes, C. L., et al., "Cationic liposomes(lipofectin) mediate retroviral infection in the absence of specificreceptors," J. Virol. (1990) 64:957-961). In contrast, for the firsttime in the art, the present studies have shown that cationic liposomescomprising AAV material mediated transfection of both dividing andnon-dividing cell types. In accordance with the invention, AAVplasmid:cationic liposomes have provided a highly efficient transfectionsystem that achieved sustained, high-level expression.

Advantageously, plasmid DNA:liposome complexes can be delivered in vivo(such as by intravenous, intraperitoneal and aerosol administration)without any measurable toxicity (Philip, R., et al., "In vivo genedelivery: Efficient transfection of T lymphocytes in adult mice," J.Biol. Chem. (1993) 268:16087-16090; Stribling, R., et al., "Aerosol GeneDelivery in vivo," Proc. Natl. Acad. Sci. USA (1992) 89:11277-11281;Zhu, N., et al., "Systemic gene expression after intravenous DNAdelivery into adult mice," Science (1993) 261:209-211; Stewart, M. J.,et al., "Gene transfer in vivo with DNA-liposome complexes: Safety andacute toxicity in mice," Human Gene Therapy (1992) 3:267-275). Inaccordance with the invention, DNA concentration can be optimized toobtain maximum expression. Thus, gene transfer by use of liposomescomprising AAV material transferred AAV and transgene material into awide variety of cell types ex vivo, and is of use in vivo as well. Thesepresent results are of immense advantage to any gene therapy protocol.

As used herein and in the appended claims, the singular forms "a,""and," and "the" include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to "a formulation"includes mixtures of different formulations and reference to "the methodof treatment" includes reference to equivalent steps and methods knownto those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference to describe and disclose specificinformation for which the reference was cited in connection with.

What is claimed is:
 1. A T cell transfected by a composition for genetic manipulation, said composition comprising:a cationic liposome; and a double-stranded circular plasmid DNA vector lacking a selectable marker and encoding at least one inverted terminal repeat from adeno-associated virus, a promoter other than an adeno-associated virus promoter and a genetic sequence of interest.
 2. The transfected cell of claim 1 wherein said cell is a CD3⁺, CD4⁺, or CD8⁺ T cell.
 3. The transfected cell of claim 1 wherein said genetic sequence of interest is the gene encoding IL-2.
 4. A T cell transfected by a method of introducing a genetic sequence of interest into a host cell, said method comprising steps of;providing a composition comprising a cationic liposome; and a double-stranded circular plasmid DNA lacking a selectable marker and encoding at least one inverted terminal repeat from adeno-associated virus, a promoter other than an adeno-associated virus promoter and a genetic sequence of interest; and contacting said composition with a host cell whereby the genetic sequence of interest is introduced into the host cell.
 5. The transfected cell of claim 4 wherein the host cell is CD3⁺, CD4⁺, or CD8⁺ T cell. 